Transgenic plants with altered levels of phenolic compounds

ABSTRACT

Methods for altering levels in plants of one or more phenolic compounds that are intermediates or final products of the plant phenylpropanoid pathway are provided. One method comprises transforming a plant cell with an expression construct comprising a nucleic acid which encodes a transactivator protein comprising the myb domain of the maize “ZmMyb-IF35” protein and an activation domain. Another method comprises transforming a plant cell with an expression construct comprising a transgene which encodes an antisense ZmMyb-IF35 RNA. The present invention also relates to expression constructs and vectors used in the present methods. transformed plant cells and transgenic plants prepared according to the present methods, and the seeds of such transgenic plants.

[0001] This application claims priority to U.S. Provisional ApplicationNo. 60/274,629, filed Mar. 8, 2001.

[0002] The present invention was made, at least in part, with supportfrom the Department of Agriculture (Grant No. USDA 1999-01582)) and theNational Science Foundation (Grant No. MCB-9974474 and MCB-9896111). TheUnited States Government has certain rights in the invention.

BACKGROUND

[0003] Plants provide an almost endless variety of chemical compoundsderived from primary or secondary metabolism. Many plant secondarymetabolites are desirable. For example, some plant secondary metabolitesprovide protection against pathogens or adverse environmentalconditions, and thus have substantial agronomic importance. In addition,a number of plant secondary metabolites serve as nutraceuticalcomponents of our diet. Furthermore, certain plant secondary metaboliteshave diverse medical applications, particularly in the pharmaceuticalindustry (See, Heilmann J. and R. Bauer (1999) Functions of PlantSecondary Metabolites and their Exploitation in Biotechnology. M. Wink.Boca Raton, CRC Press LLC. 3:274-310).

[0004] The accumulation of certain secondary metabolites in plants,however, can also be problematic. For example, the presence in trees oflarge amounts of lignin, a product of the plant phenylpropanoid pathway,can increase the costs and time required to make high quality paper.Large amounts of lignin in grasses can decrease their digestibility. Inflour products, high levels of colored pigments, which are also productsof the phenylpropanoid pathway, can make the flour products lessdesirable to the consumer.

[0005] Plant secondary metabolites can be grouped into several majorclasses including the phenolics, alkaloids, and isoprenoids. The aminoacids phenylalanine and tyrosine serve as precursors for phenoliccompounds that are intermediates or final products of a branch of thephenylpropanoid pathway. A schematic representation of theplenylpropanoid pathway which leads from phenylalanine through severalbranches to the hydroxy cinnamates, lignins, and the flavonoids is shownin FIG. 1. The phenylpropanoids, and their derivatives, and theflavonoids, and their derivatives, are examples of intermediates andfinal products of the phenylpropanoid pathway respectively. Flavonoidsare phenolic natural products that have multiple functions in plants,including roles as floral pigments for the attraction of pollinators,signaling molecules for beneficial microorganisms in the rhizosphere,and antimicrobial defense compounds. In addition, flavonoids areemerging as important nutraceuticals because of their strong antioxidantproperties, and several flavonoids show anti-tumor activities.Chlorogenic acid, another phenolic compound that is believed to be thefinal product of one branch of the phenylpropanoid pathway hasanti-pathogenic activity and bactericidal activity in plant andanti-tumor activity in animals.

[0006] The first committed step in the phenylpropanoid pathway iscatalyzed by phenylalanine ammonia lyase (PAL), which convertsphenylalanine to cinnamic acid (or tyrosine to ρ-coumaric acid in somemonocots). Transcriptional activation of genes encoding enzymes involvedin phenylpropanoid metabolism, such as PAL, 4-coumarate CoA ligase(4CL), and cinnamyl alcohol dehydrogenase (CAD), represents a key stepin the regulation of the phenylpropanoid pathway. The coordinateregulation of the PAL, 4CL and CAD genes in many plant species suggeststhe existence of specific transcription factors or transactivators thatcoordinately activate the expression of these genes.

[0007] The regulation of flavonoid biosynthesis provides the bestdescribed example of how certain transcription factors control theexpression of biosynthetic genes (reviewed in Mol et al. 41). In maize,two classes of regulatory proteins control accumulation of theanthocyanins which are flavonoid derivatives. These two classes are aMyb-domain containing class (encoded by the CI and P1 genes) and a basichelix-loop-helix (bHLH)-domain containing class (members of the RIB genefamilies). Anthocyanin production requires the interaction between amember of the Myb-domain C1/P1 family and a member of the bHLH-domainR/B family 41, and the pattern of anthocyanin pigmentation in anyparticular plant part is controlled by the combinatorial,tissue-specific expression of these regulatory genes. Orthologs, asdefined by Fitch, of the maize C1 and R regulators have been identifiedin other plants, such as petunia and snapdragon, and these regulatoryproteins have been shown to be exchangeable between monocots and dicots.

[0008] In addition to 3-hydroxy flavonoids and anthocyanins, maize andits close relatives like sorghum accumulate 3-deoxy flavonoids andderived pigments, which include the phlobaphenes. A single knowntranscription factor (P) controls 3-deoxy flavonoid and phlobaphenebiosynthesis in maize. P regulates the accumulation of a subset offlavonoid biosynthetic gene products, namely C2 (a chalcone synthase)and A1 (dihydroflavonol 4-reductase). On the basis of these and otherstudies, it is quite clear that transcription factors are importanttools for controlling the levels of flavonoids in plants.

[0009] In view of the important role of phenolic compounds that areintermediates and final products of the plant phenylpropanoid pathway,it is desirable to have additional transcription factors which arecapable of regulating the levels of these secondary metabolites inplants. Such transactivators would serve as important tools forincreasing pathogen resistance, altering digestibility, and manipulatinglevels of nutraceutical compounds, such as flavonoids and other phenoliccompounds, in plants.

SUMMARY OF THE INVENTION

[0010] The present invention provides methods for altering levels inplants of one or more phenolic compounds that are intermediates or finalproducts of the plant phenylpropanoid pathway. One method comprisestransforming a plant cell with an expression construct, hereinafterreferred to as the “ZmMyb-IF35 sense construct” comprising a DNAmolecule or transgene comprising a sequence which encodes atransactivator protein comprising the myb domain of a protein referredto hereinafter as the maize “ZmMyb-IF35” protein or a functionalequivalent thereof and an activation domain; and regenerating atransgenic plant from the transformed plant cell. The transgene furthercomprises a promoter operably linked to the transactivator proteinencoding sequence. In one embodiment, the transactivator proteincomprises the activation domain as well as the myb domain of the maizeZmMYB-IF35 protein. Another method comprises transforming a plant cellwith an expression construct, hereinafter referred to as the “ZmMyb-IF35antisense construct” comprising a DNA molecule or transgene comprising asequence which encodes an antisense ZmMyb-IF35 RNA and a promoteroperably linked to the antisense ZmMyb-IF35 RNA coding sequence; andregenerating a transgenic plant from the transformed plant cell. Theantisense ZmMyb-IF35 RNA has a sequence with sufficient complementarityto the wild-type maize ZmMyb-IF35 protein encoding sequence to preventits translation or to ensure the degradation of the sense ZmMyb-IF35 RNAin the cell. Another method comprises transforming a plant cell with anexpression construct, hereinafter referred to as the “ZmMyb-IF35 dsRNAiconstruct” comprising a DNA molecule or transgence comprising a sequencewhich encodes a ZmMyb-IF35 sense RNA coding sequence and a ZymMyb-IF35antisense RNA coding sequence, a linker sequence which links theZmMyb-IF35 sense RNA coding sequence to the ZmMyb-IF35 antisense RNAcoding sequence, and a promoter operably linked to the ZmMyb-IF35 senseRNA coding sequence and the ZmMyb-IF35 antisense RNA coding sequence;and regenerating a transgenic plant from the transformed plant cell.

[0011] The present invention also relates to a method of altering levelsin a plant of one or more phenolic compounds that are intermediates or afinal product of the phenylpropanoid pathway by introducing into suchcells an RNA molecule comprising a sequence which encodes atransactivator protein comprising the myb domain of the maize ZmMyb-IF35protein or a functional equivalent thereof and an activation domain; andexpressing the transactivator protein in the cell.

[0012] The present invention also relates to the ZmMyb-IF35 sense,antisense, and dsRNAi expression constructs and vectors which are usedin the present methods. The present invention also relates totransformed plant cells and transgenic plants prepared according to thepresent methods, and the seeds of such transgenic plants. Suchtransgenic plants comprise altered levels of one or more phenoliccompounds that are intermediates of final products of thephenylpropanoid pathway.

BRIEF DESCRIPTION OF THE FIGURES

[0013]FIG. 1 is a schematic representation of the phenylpropanoid andterpenoid indole alkaloid biosynthetic pathways. Only the core enzymesof each pathway are shown, and the names of classes of intermediates orfinal compounds are indicated. Compounds and enzymes from primarymetabolism are shown in regular type. Compounds and enzymes involved inthe secondary metabolic pathway are shown in bold type.

[0014]FIG. 2 is a schematic representation of the regulation of themaize anthocyanin biosynthetic pathways by the transcription factors Pand C1/PI+R/B.

[0015]FIG. 3 shows the nucleotide sequence, SEQ ID NO. 1, of a genomicDNA molecule which encodes the maize ZmMyb-IF35 protein. The sequencecomprises the nucleotide sequences of a portion of the 5' untranslatedregion, exon 1, intron 1, exon 2, intron 2, and exon 3 of the maizeZmMyb-IF 35 gene.

[0016]FIG. 4 shows the deduced amino acid sequence, SEQ ID NO. 2 of themaize ZmMyb-IF 35 protein.

[0017]FIG. 5 is an alignment of an allele (Prr) which encode the maizetranscription factor P and the maize ZmMyb-IF35 gene.

[0018]FIG. 6 shows the steps involved in obtaining transgenicArabidopsis plant cells lines, plants, and seeds which comprise atransgene comprising a sequence which encodes the ZmMyb-IF35 proteinoperably linked to a CaMV 35S promoter.

[0019]FIG. 7(a) shows the pigments produced in transgenic Arabidopsisseedlings which comprise a transgene comprising a sequence which encodesthe ZmMyb-IF35 protein operably linked to a CaMV 35S promoter.

[0020]FIG. 7(b) shows the absence of pigments in seeds derived fromtransgenic Arabidopsis plants which comprise a transgene comprising asequence which encodes the ZmMyb-IF35 protein operably linked to a CaMV35S promoter.

[0021]FIG. 8 shows the expression of green fluorescence in a callus ofmaize cells transformed with a transgene comprising a sequence whichencodes the maize ZmMyb-IF35 protein operably linked to a CaMV 35Spromoter.

[0022]FIG. 9 shows the accumulation of ferulic acid and chlorogenic acidin a callus of maize cells transformed with a transgene comprising asequence which encodes the maize ZmMyb-IF35 protein operably linked to aCaMV 35S promoter.

DETAILED DESCRIPTION OF THE INVENTION

[0023] Definitions

[0024] Antisense—As used herein refers to a single-stranded nucleicacid, typically RNA, having a complementary base sequence to the basesequence of a messenger RNA (mRNA).

[0025] Complementary—As used herein refers to a nucleotide sequence thatis related to another nucleotide sequence by the Watson-Crickbase-pairing rules, i.e., the sequence A-T-G-C in a DNA strand iscomplementary to the sequence T-A-C-G in a second DNA strand and to thesequence U-A-C-G in an RNA strand.

[0026] Double-stranded RNA (dsRNA)—As used herein refers topolyribonucleotide structure formed by either a singleself-complementary RNA strand or by at least two complementary RNAstrands. The degree of complementary need not necessarily be 100percent. Rather, it must be sufficient to allow the formation of adouble-stranded structure under the conditions employed.

[0027] Gene expression or expression—As used herein refers to thepresence of an RNA transcribed from a gene or a protein translated froman RNA transcribed from the gene within a call, tissue, or organism.More specifically, gene expression can be evaluated with respect to RNAexpression or protein expression. The term “gene expression” is alsoused to refer to the process by which RNA is transcribed from a gene orby which RNA transcribed from a gene is translated.

[0028] Sense—As used herein, refers to a base sequence as present in amessenger RNA (mRNA).

[0029] Vector—As used herein, refers to a nucleic acid molecule capableof mediating introduction of another nucleic acid to which it has beenlinked into a cell. One type of preferred vector is an episome, i.e., anucleic acid capable of extrachromosomal replication. Preferred vectorsare those capable of extra-chromosomal replication and/or expression ofnucleic acids to which they are linked in a host cell. Vectors capableof directing the expression of inserted DNA sequences are referred toherein as “expression vectors” and may include plasmids or viruses, inparticular baculoviruses. However, the invention is intended to includesuch other forms of expression vector which serve equivalent functionsand which become known in the art subsequently hereto.

[0030] The present invention provides sense, antisense, and dsRNAiexpression constructs which can be used to prepare transgenic plantcells, plant parts, and plants having elevated or depressed levels ofone or more phenolic compounds that are intermediates or final productsof the phenylpropanoid pathway. One example of a phenolic compound thatis believed to be the final product of one branch of the phenylpropanoidpathway is chlorogenic acid. It is also believed that coumaryl coA (SeeFIG. 1) is a precursor required for chlorogenic acid formation.Chlorogenic acid has anti-fungal activity and bactericidal activity inplants and anti-tumor activity in animals. An example of a phenoliccompound that is believed to be an intermediate in the lignin branch ofthe phenylpropanoid pathway is ferulic acid.

[0031] The ZmMyb-IF35 sense construct comprises a DNA moleculecomprising a sequence that encodes a transactivator protein comprisingthe myb domain of the maize ZmMyb-IF35 protein, or a functionalequivalent thereof, and a transactivation domain. The sense constructfurther comprises a promoter operably linked to the transactivatorprotein encoding sequence. Nucleic acid is “operably linked” when it isplaced into a functional relationship with another nucleic acidsequence. For example, a promoter or enhancer is operably linked to acoding sequence if it affects the transcription of the sequence; or aribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are covalently linkedcontiguous. However, enhancers do not have to be contiguous. Linking isaccomplished by ligation at convenient restriction sites. If such sitesdo not exist, synthetic oligonucleotide adapters or linkers are used inaccordance with conventional practice.

[0032] Another sense construct useful for altering the levels in plantcells of phenolic compounds that are intermediates or final products inthe phenylpropanoid pathway comprises an RNA molecule comprising asequence which encodes a transactivator protein comprising the mybdomain of the maize ZmMyb-IF35 protein, or a functional equivalentthereof, and a transactivation domain. Such RNA molecule is operablylinked to regulatory sequences which permit translation of the RNAmolecule within plant host cells.

[0033] The ZmMyb-IF35 antisense construct comprises a DNA moleculecomprising a sequence which encodes an antisense RNA molecule comprisinga sequence with sufficient complementarity to the maize ZmMyb-IF35 RNAcoding sequence to stably bind thereto, and thus, prevent itstranslation. The antisense RNA comprises at least 15 nucleotides. TheZmMyb-IF35 antisense construct further comprises a promoter operablylinked to the antisense RNA encoding sequence.

[0034] The ZmMyb-IF35 dsRNAi construct comprises a DNA molecule ortransgence comprising a sequence which encodes a sense ZmMyb-IF35 RNAcoding sequence and an antisense ZmMyb-IF35 RNA coding sequence and alinker sequence which links the sense ZmMyb-IF35 RNA coding sequence tothe antisense ZmMyb-IF35 RNA coding sequence. The linker is at least 300in length, and preferably, from 400 to 500 base pairs in length. Oneexample of such a linker is a 400 bp from ff in the bacterial UdAencoding GUS. The transgene is operably linked to a promoter whichdrives expression of the sense ZmMyb-IF35 RNA and the antisenseZmMyb-IF35 RNA. The double-stranded RNA that results from expression ofthis construct prevents accumulation of ZmzMyb-IF35 mRNA through apost-transcription gene silencing method known in the art asdouble-stranded RNA interference.

[0035] ZmMyb-IF35

[0036] As used herein the term “ZmMyb-IF35” refers to a maize Mybprotein which has a molecular weight of about 38 kDa and comprises an Nterminal R2R3 myb domain. In one embodiment, the maize ZmMyb-IF35protein has the amino acid sequence, SEQ ID NO. 2, shown in FIG. 4. Thenucleotide sequence, SEQ ID NO. 1, of a genomic DNA molecule whichencodes this form of the maize ZmMyb-IF35 protein is shown in FIG. 3.The 5′ untranslated region (5'UTR) of this DNA molecule extends fromnucleotide 1 through 197 of SEQ ID NO. 1. The first exon sequenceextends from nucleotide 198 through 321; the second exon sequenceextends from nucleotide 415 through 544, and the third sequence extendsfrom nucleotide 2736 through nucleotide 3564. The nucleotide sequenceencoding the myb domain of maize ZmMyb-IF35 begins at nucleotide 232within exon 1, encompasses all of exon 2, and ends at nucleotide 2817within exon 3.

[0037] The amino acid sequence of the Myb domain of maize ZmMyb-IF35,which extends from amino acid 12 through amino acid 115 in SEQ ID NO. 2,has 86% identity to the amino acid sequence of the Myb domain of themaize P protein. Outside of the Myb domain, the amino acid sequence ofZmMyb-IF35 has only 33% identity with the amino acid sequence of themaize P protein. The % of amino acid identity was determined usingCluatal-W formatted Alignments.

[0038] Transactivator Protein

[0039] The transgene encoding the transactivator protein comprises asequence which encodes the maize ZmMyb-IF35 myb domain or a functionalequivalent thereof and a sequence which encodes an activation domain.The term “functional equivalent” as used herein refers to a polypeptidewhose amino acid sequence is at least 95% identical, preferably 97%identical, more preferably at least 99%, identical to the amino acidsequence which includes and extends from amino acid 12 through aminoacid 115 of SEQ ID NO. 2. Such functional equivalents when linked to anactivation domain and incorporated into a maize cell enhance productionof chlorogenic acid in the resulting transformed maize host cells in thesame manner and to the same extent as the naturally-occurring maizeZmMyb-IF35 protein. Levels of chlorogenic acid in the transformed hostcells are assayed using standard techniques such as high performanceliquid chromatography (HPLC).

[0040] Such functional variants have an altered sequence in which one ormore of the amino acids is deleted or substituted, or one or more aminoacids are inserted, as compared to the reference amino acid sequence,i.e., amino acid 12 through amino acid 115 of SEQ. ID. NO.:2. Sequenceswhich are at least 95% identical have no more than 5 alterations, i.e.any combination of deletions, insertions or substitutions, per 100 aminoacids of the reference amino acid sequence. Percent identity may bedetermined by comparing the amino acid sequence of the functionalvariant with the reference sequence, i.e. the sequence extending fromand including amino acid 12 through amino acid 112 in SEQ ID NO. 2 usingMEGALIGN project in the DNA STAR program. The variant sequences andreference sequences are aligned for identity calculations using themethod of the software basic local alignment search tool in the BLASTnetwork service (the National Center for Biotechnology Information,Bethesda, MD) which employs the method of Altschul, S. F., Madden, T.L., Shäffer, A. A., Zhang, J., Zhang, Z., and Miller, W. (1997) NucleicAcid Res. 25, 3389-3402. Identities are calculated, for example, by theAlign program (DNAstar, Inc.) In all cases, internal gaps and amino acidinsertions in the candidate sequence as aligned are not ignored whenmaking the identity calculation. Preferably, the substitutions,deletions, or additions are made at the positions marked with an X inthe maize ZmMyb-IF35 myb domain sequence, SEQ ID NO. 2, shown below.

Mvb Domain Amino Acid Sequence of IF35

[0041] LKXGRWTXEEDXXLAXYIXEHGEGSWRSLPKNAGLLRCGKSCRLRWINYLRAXXKRGNIXXEEEDXIXKLHATLGNRWSLIAXHLPGRTDNEIKNYWNSHLSRX

[0042] While functional variants of the myb domain of the maize ZmMyb-IF35 protein may have non-conservative amino acid substitutions, it ispreferred that the functional variant have the conservative amino acidsubstitutions. In conservative amino acid substitutions, the substitutedamino acid has similar structural or chemical properties with thecorresponding amino acid in the reference sequence. By way of example,conservative amino acid substitutions involve substitution of onealiphatic or hydrophobic amino acids, e.g. alanine, valine, leucine andisoleucine, with another; substitution of one hydroxyl-containing aminoacid, e.g. serine and threonine, with another; substitution of oneacidic residue, e.g. glutamic acid or aspartic acid, with another;replacement of one amide-containing residue, e.g. asparagine andglutamine, with another; replacement of one aromatic residue, e.g.phenylalanine and tyrosine, with another; replacement of one basicresidue, e.g. lysine, arginine and histidine, with another; andreplacement of one small amino acid, e.g., alanine, serine, threonine,methionine, and glycine, with another.

[0043] The term “activation domain” as used herein refers to a peptidewhich allows the transactivator protein to interact with an RNApolymerase or with components of the basal transcription machinery. Theactivation domain may be directly linked by a peptide bond to the Cterminus of the ZmMyb-IF35 myb domain or its functional equivalent.Alternatively, there may be a linker comprising from 1 to 500 aminoacids between the C terminus of the ZmMyb-IF35 myb domain or itsfunctional equivalent and the N terminus of the activation domain. Theactivation domain may have a sequence which is at least 95% identical,preferably 97% identical, more preferably at least 99%, identical to theamino acid sequence which includes and extends from amino acid 116through amino acid 345 of SEQ ID NO. 2. Alternatively the activationdomain may be derived from another myb protein. Examples of othersources for the activation domain include, but are not limited to, themaize C1 regulator protein, the yeast Ga14 protein, and the VP16 proteinfrom herpes virus.

[0044] Examples of nucleotide sequences which encode the transactivatorproteins of the present invention include, but are not limited to, asequence which encodes amino acid 1 through amino acid 345 of SEQ ID NO.2, a sequence which comprises the sequences of the first, second andthird exons of SEQ ID NO. 1, and a sequence which binds under highlystringent hybridization conditions to a sequence which comprisesnucleotide 232 through nucleotide 331, nucleotide 415 through nucleotide544, and nucleotide 2736 through nucleotide 3564 of SEQ ID NO. 1.Hybridization conditions are based on the melting temperature TM of thenucleic acid binding complex or probe, as described in Berger and Kimmel(1987) Guide to Molecular Cloning Techniques, Methods in Enzymology,Vol. 152, Academic Press. The term “stringent conditions”, as usedherein, is the “stringency” which occurs within a range from about Tm-5(5° below the melting temperature of the probe) to about 20° C. belowTm. “Highly Stringent hybridization conditions” refers to an overnightincubation at 42 degree C in a solution comprising 50% formamide, 5×SSC(750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.2×SSC atabout 65 degree C for 30 minutes. As recognized in the art, stringencyconditions can be attained by varying a number of factors such as thelength and nature, i.e., DNA or RNA, of the probe; the length and natureof the target sequence, the concentration of the salts and othercomponents, such as formamide, dextran sulfate, and polyethylene glycol,of the hybridization solution. All of these factors may be varied togenerate conditions of stringency which are equivalent to the conditionslisted above.

[0045] Construct

[0046] The ZmMyb-IF35 sense, antisense, and dsRNAi expression constructsfurther comprise a promoter which is operably linked to thetransactivator protein encoding sequence or the ZmMyb-IF35 antisense RNAencoding sequence, or the ZmMyb-IF-35 antisense RNA and sense RNA codingsequences, respectively. The promoter may be constitutive, inducible ortissue specific promoter.

[0047] The promoters may be obtained from genomic DNA by usingpolymerase chain reaction (PCR), and then cloned into the construct.Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by J. Sambrook, E. F. Fritschand T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1989) and by T. J. Silhavy,M. L. Berman, and L. W. Enquist, Experiments with Gene Fusions, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984).

[0048] Optionally, the ZmMyb-IF35 sense and antisense contructs furthercomprise a transcriptional terminator which is operably linked to thetransactivator protein encoding sequence or the ZmMyb-IF 35 antisenseRNA sequence, respectively. A variety of transcriptional terminators areavailable for use in the expression constructs. These are responsiblefor the termination of transcription beyond the transgene and itscorrect polyadenylation. Appropriate transcriptional terminators andthose which are known to function in plants include the CaMV 35Sterminator, the tm1 terminator, the nopaline synthase terminator and thepea rbcS E9 terminator.

[0049] Optionally, the construct further comprises sequences for theenhancement or regulation of expression. Numerous sequences have beenfound to enhance gene expression from within the transcriptional unitand these sequences can be used in conjunction with the DNA molecules ofthis invention to increase their expression in transgenic plants.Various intron sequences have been shown to enhance expression,particularly in monocotyledonous plants. For example, the introns of themaize Adhl gene have been found to significantly enhance the expressionof the wild-type gene under control of its cognate promoter whenintroduced into maize cells. Intron 1 was found to be particularlyeffective and enhanced expression in fusion constructs with thechloramphenicol acetyltransferase gene (Callis et. al., Genes Develop.1:1183-1200, 1987). In the same experimental system, the intron from themaize bronze 1 gene had a similar effect in enhancing expression (Calliset al., supra). Intron sequences have been routinely incorporated intoplant transformation vectors, typically within the non-translatedleader.

[0050] A number of non-translated leader sequences, derived fromviruses, are also known to enhance expression. Specifically, leadersequences from Tobacco Mosaic Virus (TMV, the “omega sequence”), MaizeChlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AlMV) have beenshown to be effective in enhancing expression (e.g., Gallie et. al.,Nucl. Acids Res. 15:8693-8711, 1987; Skuzeski et. al., Plant Mol. Biol.15:65-79, 1990).

[0051] The constructs of the present invention are synthesized byinsertion of a sequence comprising the transactivator protein encodingsequence or the antisense RNA encoding sequence or both into an emptyexpression cassette. Such empty expression cassettes, providingappropriate regulatory sequences for plant expression of the encodingsequence are well-known. To produce the transactivator protein, theprotein encoding sequence is placed in correct orientation in theconstruct. The transgene preferably employs plant-preferred codons toenhance expression of the transgene. To produce an antisense mRNA whichcan interfere with indigenous production of the ZmMyb-IF35 protein, theZmMyb-IF35 protein encoding sequence is placed in the expressionconstruct in the opposite orientation. The nucleotide sequence of thetransgene, either DNA or RNA, can readily be derived from the amino acidsequence for the protein using standard texts. The transgene preferablyemploys plant-preferred codons to enhance expression of the transgene.

[0052] The present invention also provides a vector, such as for examplea plasmid, which comprises the expression construct The term “vector” asused herein refers to DNA molecules which are able to replicate and toexpress a foreign gene in a host cell. Typically, the vector has one ormore restriction endonuclease recognition sites which permit insertionof the expression construct or transgene into the vector. Preferably,the vector further comprises a marker gene, such as for example, adominant herbicide resistance gene or antibiotic resistance gene, whichencode compounds that serve to identify and separate transformed cellsfrom non-transformed cells. Examples of suitable marker genes includethe bar gene which codes for phosphinothricin acetyl transferase, akanamycin resistance gene, and a hygromycin resistance gene. A cell inwhich the foreign genetic material in the vector is functionallyexpressed has been “transformed” by the vector and is referred to as a“transformant”. Expression of the transactivator protein encodingsequence and the antisense RNA encoding sequence in transformants may bemonitored using Northern blot techniques.

[0053] Optionally, the vector may include partial T-DNA bordersequences, typically retained on integrated DNA following a T-DNAinsertion event. Alternately, the integrated exogenous DNA may show sometruncation of the left end of the T-DNA, or occasionally, of some DNAbeyond the left border, as has been observed after transformation withAgrobacterium.

[0054] Vectors suitable for transforming plant cells include, but arenot limited to, Ti plasmids from Agrobacterium tumefaciens (J. Darnell,H. F. Lodish and D. Baltimore, Molecular Cell Biology, 2nd edition,Scientific American Books, N.Y. (1990)), a plasmid containing aβ-glucuronidase gene and a cauliflower mosaic virus (CaMV) promoter plusa leader sequence from alfalfa mosaic virus (Sanford et. al., Plant Mol.Biol. 22:751-765, 1993) or a plasmid containing a bar gene cloneddownstream from a CaMV 35S promoter and a tobacco mosaic virus (TMV)leader. Other plasmids may additionally contain introns, such as thatderived from alcohol (Adhl), or other DNA sequences. The size of thevector is not a limiting factor.

[0055] Transformation of Plant Cells

[0056] Any type or source of plant cells which can serve as a target fortransformation by any one or more of the various biological andnon-biological delivery mechanisms available in the art can serve as atarget for transformation according to the present method. Theseinclude, but are not necessarily limited to, immature and matureembryos, pollen, protoplasts, suspension culture cells, callus cells,cotyledons or other seed and seedling parts, leaves or leaf pieces, androots or root pieces.

[0057] Host cells which serve as the target from transformation can bederived from monocotyledonous or dicotyledonous plants. In preferredembodiments, the host cells are obtained from maize, rice, sorghum,cotton and soybeans.

[0058] The transformed host cells are useful for preparing transgenicplants or transgenic callus lines or cell lines with altered levels ofphenolic compounds. The transformed host cells are also useful sourcesof important phenolic compounds. These compounds are extracted from thehost cells using procedures known in the art.

[0059] Methods of Transforming Plant Cells

[0060] Delivery or introduction of the ZmMyb-IF35 sense and antisenseexpression contructs into the host plant cells, may be accomplished by avariety of techniques available in the art. Such techniques includenon-biological mechanisms such as microprojectile bombardment,electroporation, microinjection, induced uptake, and aerosol beaminjection, as well as biological methods such as direct DNA uptake,liposomes and Agrobacterium-mediated transformation. See, for example,Bilang, et. al., Gene 100:247-250, 1991; Scheid et. al., Mol. Gen.Genet. 228:104-112, 1991; Guerche et. al., Plant Science 52:111-116,1987; Neuhause et. al., Theor. Appl Genet. 75:30-36, 1987; Klein et.al., Nature 327:70-73 1987; Howell et. al., Science 208:1265, 1980;Horsch et. al., Science 227:1229-1231, 1985; DeBlock et. al., PlantPhysiology 91:694-701, 1989; Methods for Plant Molecular Biology,Weissbach and Weissbach, eds., Academic Press, Inc., 1988; and Methodsin Plant Molecular Biology, Schuler and Zielinski, eds., Academic Press,Inc., 1989. See also, U.S. Pat. Nos. 4,945,050; 5,036,006; and5,100,792, all to Sanford et. al. Combinations of the above methods mayalso be used.

[0061] Transformation of host cells derived from monocotyledonousplants, preferably, is achieved using microprojectile bombardment. Asused herein “microprojectile bombardment” is used to refer to thegeneral method of delivering nucleic acids, including DNA and RNA, to aliving cell by coating or precipitating the nucleic acids onto amicroprojectile, preferably gold particles, and propelling the coatedmicroprojectile into the living cell (see e.g., U.S. Pat. No. 5,036,006issued Jul. 30, 1991 to Sanford et. al.; U.S. Pat. No., 5,302,523,issued Apr. 12, 1994 to Coffee; Vasil et. al., Biotechnology11:1553-1558, 1993; and Weeks et. al., Plant Physiol. 102:1077-1084,1993).

[0062] The exact amount of the construct provided to the host cell isnot critical and may vary depending on the manner and form in which thecomponent is delivered. If desired, the skilled artisan may routinelyvary the amount of construct delivered to determine the optimum levelfor each using a particular delivery system.

[0063] The successful delivery of the DNA or RNA construct into the hostcell may be preliminarily evaluated by the transient expression of a“reporter” gene. A reporter gene is a component on the expression vectorintroduced into the cell, or a component of a separate DNA constructwhich is co-introduced into the cell along with the DNA constructcomprising the transgene. The property conferred on the transformed cellor tissue by the introduction of the reporter gene is usually easilydetectable (e.g., expression of an easily assayable enzyme). “Transientexpression” denotes the expression of a gene before the gene has beenstably integrated into the genome of the treated cells or tissue. Forexample, commonly used reporter genes are the genes coding for theproduction of chloramphenicol acetyltransferase, which confersresistance to the antibiotic chloramphenicol, or the E.coliβ-glucuronidase gene (gusA), the products of which can be detected by ahistochemical assay.

[0064] Cells that express reporter genes in transient assays may notgive rise to cells where the transformed DNA becomes stably integratedinto the host cell genome. Selection of cells that express variousmarker genes, however, does give rise to cells in which the transformedDNA is stably integrated into the host cell genome. Herein, “selection”means conditions where only cells into which the DNA construct has beendelivered will grow and cells in which the DNA construct has not beendelivered will not grow. For example, cells stably expressing anintroduced neomycin phosphotransferase gene are selected by growth inthe drug G418. Cells stably expressing an introduced herbicideresistance gene are selected by growth in the presence of the herbicide.Shoots or plantlets growing in the presence of the drug or herbicide arepresumptively transformed. Confirmation of stable integration of thetransformed genes into the genome of the host may later be accomplishedby, for example, herbicide treatment of the resulting plants. Inaddition, later molecular detection of the introduced DNA in theisolated genomic DNA of the plant cells, for example using Southernblotting/hybridization or polymerase chain reaction, may be used toconfirm integration of the introduced genes into the genome of the host.

[0065] Transformed plant host cells are used to regenerate transgenicplants. In plants, every cell is capable of regenerating into a matureplant and, in addition, contributing to the germ line such thatsubsequent generations of the plant will contain the transgene. Growthof transformed plant cells and regeneration of such cells into matureplants is routine among those skilled in the art.

[0066] The transgenic plants are then grown and pollinated with eitherthe same transformed strain or with different strains, and the resultinghybrid, having the desired phenotypic characteristic, is identified. Twoor more generations may be grown to ensure that the desired phenotypiccharacteristic is stably maintained and inherited and then seedsharvested. Transformed progeny obtained by this method may bedistinguished from non-transformed progeny by the presence of theintroduced transgene(s) and/or accompanying DNA in the genome of theplant. Transformed plants also may be distinguished from non-transformedplants by a change in phenotype. For example, transformed plants may bedistinguished from non-transformed plants by the presence of thetransactivator protein or antisense RNA in tissues or cells where it isnot normally not present or by an increase or decrease in the amount ofthe transactivator protein in cells where it normally is present.

[0067] The present invention also encompasses plant cells which aretransiently transfected with an RNA molecule which encodes atransactivator protein comprising a ZmMyb-IF35 myb domain and anactivation domain. Such cells are useful for producing large amounts ofdesirable phenolic compounds that are intermediates or final products ofthe phenylpropanoid pathway.

EXAMPLES

[0068] The following examples are for purposes of illustration only andare not intended to limit the scope of the invention as defined in theclaims which are appended hereto. The references cited in this documentare specifically incorporated herein by reference.

Example 1

[0069] DNA constructs comprising the CaMV 35S promoter, operably linkedto a cDNA encoding a transactivator protein comprising SEQ ID NO. 2, anda herbicide resistance gene were prepared using standard recombinant DNAtechniques. All constructs were introduced into Arabidopsis cells via anAgrobacterium vector. Following infection, plants were regenerated fromthe transformed cells. (See FIG. 6.) As shown in FIG. 7(a) seedlingsderived from these transformed cell lines exhibited accumulation ofanthocyanin pigments near and around the cotyledons. The secondgeneration plants derived from cell line 10 and the third generationplants derived from line 2 also exhibited colorless seed coats (See FIG.7(b)), indicating that the expression ov ZmMyb-IF35 is interfering withthe accumulation of tannins.

Example 2

[0070] DNA constructs comprising the CaMV 35S promoter, operably linkedto a cDNA encoding a transactivator protein comprising SEQ ID NO. 2, anda herbicide resistance gene were prepared using standard recombinant DNAtechniques. All constructs were introduced into maize Black MexicanSweet cells via particle bombardment with gold particles using a DuPontBiolistic particle delivery system. After bombardment, cells weretransferred to fresh medium.

[0071] As shown in FIG. 8, the transformed maize callus cells producedin accordance with this method exhibit green fluorescence on the cellwalls, while control lines transfected with a construct containing theherbicide resistance gene but lacking a DNA which encodes the ZmMyb-IF35protein exhibited yellow bodies in the cytoplasm.

[0072] As shown if FIG. 9, the transformed maize callus cells alsocontained elevated levels of the phenolic compounds ferulic acid andchlorogenic acid.

1 2 1 3845 DNA maize ZmMyb-IF-35 gene 1 tcgacccacg cgtccgcacc agcagcagagccccgagcaa tcccttctcg cccttttcac 60 ttttcagatc cagccagcca gccagccatcccactccgca ccgcattcct cgcagtggca 120 gagcttgcag cggtggtctc ttccccttcctcctgtctcc tcctcgctcg atctcctctc 180 ccaagcagcg agaagcggat ggggagggcgccgtgctgcg agaaggtggg gctgaagaag 240 gggaggtgga ccaaggagga ggacgaggtcctggcgaggt acatcaagga gcacggggaa 300 ggatcgtgga ggtcactgcc caagaatgccggtacggatc gaagtggcga gtttgttata 360 ttagctagct ctgtgtgttg gcaataggggggctgaggct ggttttgctg gcagggctgc 420 tgcggtgcgg gaagagctgc aggctgcggtggatcaacta cctgcgggcg ggtctcaaga 480 gggggaacat ctcggaggag gaggaggacatgatcatcaa gctccacgcc acgcttggca 540 acaggtacct gtccatgcct atctatctatctactatctg gcattcgatt ccttcaacct 600 cgagctgctt gggccgtgtc cacagtccatcttttctctt tgattaattc ctgcgcttaa 660 tcaactccat ccatgctctg ggccgcttcagggttcagag ttcttgcacg gtacgtagta 720 gggggacgaa aaaccctgtg cgctttattatgccaccgac cgacagtggc gaatctaggt 780 ccaatttact tgatattttt ttttttctctctctctctcc atctggtgca aagtgagaac 840 gggagggcat gtgccaagaa gctctgttccccccttccac tgaaagagaa ggaaccaaaa 900 tcaaatggca gaggaacacg gagttgccaaaaccaggccg cgcgtagatg cccctccccc 960 tcccttcaat tgttcattca gatagagcctgtttgactag tatgctatct ttgttccatc 1020 aaaaaaaata tagatggttg aaatgtagttttagagacta gaagaggtgc ggaaaaggtc 1080 gcggttttag caatgccttc cggatgtccagttagtgtcg tggtgcaccc gccgtcttgc 1140 cggccggcta ggtagaaacg tggtcaagacacatttcttt ctagacggag acggaagagg 1200 aggaagccgc ccccccgtgt gctttcatggacaggatggt cgagcaagca aaaaccctgc 1260 acactggacg gtcacctttg tcaatggccactttttgtca tggggcgagt gagtgcacgg 1320 tacttttcta cgtccgccct cgttggcctcgtgcgcgcac gtagaatgtg ccgccatcgc 1380 catggaaaag aagagggcag ccgcagcgcagggaattatt tcttcggttt ctctttcctc 1440 cgcgcctcaa tcgttggtca ttcccattggcggattaaaa acaatcctaa gggctagttt 1500 agaaacctcg ttttcccatg agatttttatttttttaagg aaaattattc attttctctt 1560 ataaaaatag gaatccctta gaaaaatagtgttcccaaaa cactattttc taagggtttt 1620 tcgtttttcc aagataaatt agttcattttttttgaaaaa ttggaaattt catggaaaaa 1680 tggtgttggc aaactagtcc taaatctgcaactttaataa tccttcgttc tgtcattatt 1740 agtaccccta ctcgttttag ctctgtttttttttaatttg agatatagac ataaaaagaa 1800 cctaaattca gacgtctaaa caaattttaccgagttgcaa aatgaatgaa tcaggacccc 1860 cctaaattta gctctgctgc aggccgctggcaaggcatgt agggcagggc agtcgtttgc 1920 cacgcggtcc ggctcgctta acacgtggtttgaatatata ttttttactc agacacgcga 1980 tagaaaaaag atgccggagt tagggagagaaaaagaaagg ggaatattcc ttgtccagcg 2040 aagagctagg ccacacccac acgatatggactgcactgca cgtactggga tattcggtat 2100 cctggtcacc ccggcattat ttggacaatatatatatgta ggggcgggtc cgcgatccca 2160 aagtcggacg cgctacgtgt tatttggacgcctggaacct ctctcgtttc tcacgtggga 2220 ctatcgtacc cctactctac gtgtatctatatcgtgctcg tcacatgaca cgcacaccac 2280 ttgtcggtag acagacatcg gcccccaagaaccgaagtgc tacgccctct ccccgaccac 2340 tgcacactgg tgcctgtcgc actgtatgagagatgcgtgg ctcggcaaat tcggagcgga 2400 ttaatgtcgt caccaagaaa ctagaaaccacttgcgttcg tcacctttca tggaccccag 2460 cagctgcagc aatcctgcca acggaaacgcgcgcacatgg tgcattagtt cgcgtggacg 2520 ccgctgcgat ccttcatttc gtttcgtttatttactatac tcgcgcgcgc cgcagctagc 2580 tatggttgtt agatcaccag cacgcgtattgattgccaca tgtgcctgcc gcctggactg 2640 gacctgcagt gcagctcctg tcctgtgcacgcctctccct gctgttctta gtctcatcaa 2700 cctcaagttt cattcttctt ttcttctccccgcaggtggt ccctgatcgc cggtcacttg 2760 cccggtcgaa cagacaacga gatcaaaaactactggaact cgcacctgag caggcgggcg 2820 gccgacttcc gcgacggcgt cgtcgtcgacatcgacctca gcaagctgcc cggcggcggg 2880 aaacggcgcg gcggccgggc cagccggggcgccgtcgtgg ccgcggccaa ggagaagaag 2940 gccaaggaga aggacgacag gggcaatagcaaggtcgcag aagcggagca gcagctcagg 3000 gacacggagg acgacgacgg cggcagcgtctccacgccga ggcctcagtc tgatgactgc 3060 ggcaccgccc agagcgaaga ggagcaagcgcaggccagcg ccagcggcct gacatccgat 3120 gggcatgggc ccgaggagga ggaggaggaggacccgctgg ctctgagcga ggagatggtg 3180 agtgcgcttc tggccccgga aagcccaaagctggaggtgg gccccgatgg ctcgtgcatg 3240 gacagctaca gtggccctcc gtcaggggaaagcggctgtg ggtccagtgg gccttctggc 3300 gacgtggccc aggacctgga cctagacgacgacaaggcca tcatggactg ggacttgatg 3360 gggctgggac atctcgaccc gccggtgacatgtgggacca gctggtgtgg gactacgacg 3420 aaacgttggt cacggaaccg gaaggaggggaggaagggca ccagcagcag gacgatgtca 3480 tgtcagacct cttcttcctg gacaatctctaggaggtgcg aggatagcat gggcatggct 3540 gccgtgatgc tttatgcttt ttaatttgatccggtacttg taggtttttg ggtgtgttca 3600 gttcaaagat gagtggcggt gtcagagacgagataagggg agtgctccag tgacatcttt 3660 gtttgctggc cggatctcac gaacccgtagaatggcaaga atgtagaaaa ataagcacgc 3720 aatatcactt ggaaaccttt catcagtagagcctgtctaa catctacaga cggagaaatg 3780 caaaaaaaaa aaaaaaaggt tgctggggttataaaaaaaa aaaaaaaaaa aaaaaaaaaa 3840 aaaaa 3845 2 363 PRT maizeZmMyb-IF-35 protein 2 Met Gly Arg Ala Pro Cys Cys Glu Lys Val Gly LeuLys Lys Gly Arg 1 5 10 15 Trp Thr Lys Glu Glu Asp Glu Val Leu Ala ArgTyr Ile Lys Glu His 20 25 30 Gly Glu Gly Ser Trp Arg Ser Leu Pro Lys AsnAla Gly Leu Leu Arg 35 40 45 Cys Gly Lys Ser Cys Arg Leu Arg Trp Ile AsnTyr Leu Arg Ala Gly 50 55 60 Leu Lys Arg Gly Asn Ile Ser Glu Glu Glu GluAsp Met Ile Ile Lys 65 70 75 80 Leu His Ala Thr Leu Gly Asn Arg Trp SerLeu Ile Ala Gly His Leu 85 90 95 Pro Gly Arg Thr Asp Asn Glu Ile Lys AsnTyr Trp Asn Ser His Leu 100 105 110 Ser Arg Arg Ala Ala Asp Phe Arg AspGly Val Val Val Asp Ile Asp 115 120 125 Leu Ser Lys Leu Pro Gly Gly GlyLys Arg Arg Gly Gly Arg Ala Ser 130 135 140 Arg Gly Ala Val Val Ala AlaAla Lys Glu Lys Lys Ala Lys Glu Lys 145 150 155 160 Asp Asp Arg Gly AsnSer Lys Val Ala Glu Ala Glu Gln Gln Leu Arg 165 170 175 Asp Thr Glu AspAsp Asp Gly Gly Ser Val Ser Thr Pro Arg Pro Gln 180 185 190 Ser Asp AspCys Gly Thr Ala Gln Ser Glu Glu Glu Gln Ala Gln Ala 195 200 205 Ser AlaSer Gly Leu Thr Ser Asp Gly His Gly Pro Glu Glu Glu Glu 210 215 220 GluGlu Asp Pro Leu Ala Leu Ser Glu Glu Met Val Ser Ala Leu Leu 225 230 235240 Ala Pro Glu Ser Pro Lys Leu Glu Val Gly Pro Asp Gly Ser Cys Met 245250 255 Asp Ser Tyr Ser Gly Pro Pro Ser Gly Glu Ser Gly Cys Gly Ser Ser260 265 270 Gly Pro Ser Gly Asp Val Ala Gln Asp Leu Asp Leu Asp Asp AspLys 275 280 285 Ala Ile Met Asp Trp Asp Leu Met Gly Leu Gly His Leu AspPro Pro 290 295 300 Val Thr Cys Gly Thr Ser Trp Cys Gly Thr Thr Thr LysArg Trp Ser 305 310 315 320 Arg Asn Arg Lys Glu Gly Arg Lys Gly Thr SerSer Arg Thr Met Ser 325 330 335 Cys Gln Thr Ser Ser Ser Trp Thr Ile SerArg Arg Cys Glu Asp Ser 340 345 350 Met Gly Met Ala Ala Val Met Leu TyrAla Phe 355 360

What is claimed is:
 1. An expression construct for preparing plant cellswith altered levels of one or more phenolic compounds that areintermediates or final products of the plant phenylpropanoid pathway,comprising: a) a nucleic acid comprising a sequence which encodes atransactivator protein comprising the maize ZmMyb-IF35 myb domain or afunctional equivalent thereof, and an activation domain; b) a promoterfor regulating transcription of said nucleic acid, said promoter beingoperably linked to said transactivator protein encoding sequence.
 2. Theconstruct of claim 1 wherein the sequence of the ZmMyb-IF35 myb domainis at least 95% identical to a sequence comprising amino acid 12 throughamino acid 115 of SEQ ID NO.
 2. 3. The construct of claim 1 wherein thesequence of the ZmMyb-IF35 myb domain is at least 97% identical to asequence comprising amino acid 12 through amino acid 115 of SEQ ID NO.2.
 4. The construct of claim 1 wherein the sequence of the ZmMyb-IF35myb domain is at least 99% identical to a sequence comprising amino acid12 through amino acid 115 of SEQ ID NO. 2
 5. The construct of claim 1wherein the sequence of said activation domain is at least 95% identicalto a sequence comprising amino acid 116 through amino acid 345 of SEQ IDNO.
 2. 6. The construct of claim 1 wherein the sequence of saidactivation domain is at least 99% identical to a sequence comprisingamino acid 116 through amino acid 345 of SEQ ID NO.
 2. 7. The constructof claim 1 wherein the sequence of said transactivator protein comprisesamino acid 116 through amino acid 345 of SEQ ID NO.
 2. 8. The constructof claim 1 wherein the transactivator protein encoding sequence bindsunder highly stringent hybridization conditions to a sequence whichcomprises nucleotide 232 through nucleotide 331, nucleotide 415 throughnucleotide 544, and nucleotide 2736 through nucleotide 3564 of SEQ IDNO.
 1. 9. The construct of claim 1 wherein the promoter is an induciblepromoter.
 10. The construct of claim 1 wherein the promoter is a tissuespecific promoter
 11. The construct of claim 1 wherein the promoter is aconstitutive promoter.
 12. A method of preparing a plant cell withaltered levels of one or more phenolic compounds, comprising:introducing the DNA construct of claim 1 into the plant cell, andmaintaining the plant cell under conditions which permit expression ofsaid transactivator protein.
 13. A plant cell prepared according to themethod of claim
 12. 14. The plant cell of claim 13 wherein the plantcell is derived from a plant selected from the group consisting ofmaize, sorghum, rice, cotton and soybean.
 15. A method of preparing atransgenic plant containing altered levels of one or more phenoliccompounds, comprising a) transforming a plant cell with the expressionconstruct of claim 1; and b) regenerating a plant from said transformedplant cell.
 16. A transgenic plant prepared by the method of claim 15.17. An expression construct for preparing a plant with altered levels ofone or more phenolic compounds of the phenylpropanid pathway of theplant comprising: a) a DNA molecule comprising a sequence which encodesan antisense RNA comprising a sequence which is complementary to atleast 115 consecutive nucleotides in an RNA that encodes SEQ ID NO.2.;and b) a promoter for regulating transcription of said DNA molecule,said promoter being operably linked to said antisense RNA encodingsequence.
 18. A method of preparing a transgenic plant containingaltered levels of one or more phenolic compounds of the phenylpropanidpathway of the plant, comprising a) transforming a plant cell with theexpression construct of claim 17; and b) regenerating a plant from saidtransformed plant cell.
 19. A transgenic plant prepared by the method ofclaim
 18. 20. An expression construct that, when contained in a plantcell, generates RNA which is sufficiently complementary to an RNAtranscribed from an endogenous ZmMyb-IF35 gene to prevent synthesis ofendogenous ZmMyb-IF35 protein in the plant cell, wherein said expressionconstruct comprises the reverse transcript of said complementary RNAoperably linked to a promoter which effects its transcription into saidcomplementary RNA, and wherein said endogenous ZmMyb-IF35 proteincomprises a sequence which comprises amino acid 1 through amino acid 345of SEQ ID NO.
 2. 21. Isolated DNA or RNA comprising a sequence which (a)encodes amino acid 1 through amino acid 345 of SEQ ID NO. 2; or (b)comprises nucleotide 232 through nucleotide 331, nucleotide 415 throughnucleotide 544, and nucleotide 2736 through nucleotide 3564 of SEQ IDNO. 1; or (c) is the complement of the amino acid encoding sequence of(a); or (d) is the complement of the sequence of (b); or (e) binds underhighly stringent hybridization conditions to a sequence which comprisesnucleotide 232 through nucleotide 331, nucleotide 415 through nucleotide544, and nucleotide 2736 through nucleotide 3564 of SEQ ID NO. 1; or (f)is the complement of (e).
 22. A transgenic plant, plant part, plantcell, or tissue culture, each of which is transformed with an isolatednucleic acid comprising a sequence selected from the group consisting of(a) a sequence comprising nucleotide 232 through nucleotide 331,nucleotide 415 through nucleotide 544, and nucleotide 2736 throughnucleotide 3564 of SEQ ID NO. 1 or its complement; (b) a sequence whichencodes amino acid 1 through amino acid 245 of SEQ ID NO. 2 or itscomplement; and (c) a sequence which binds under highly stringenthybridization conditions to a sequence which comprises nucleotide 232through nucleotide 331, nucleotide 415 through nucleotide 544, andnucleotide 2736 through nucleotide 3564 of SEQ ID NO.
 1. 23. Anexpression construct for preparing a plant with altered levels of one ormore phenolic compounds of the phenylpropanid pathway of the plantcomprising a) a DNA molecule comprising a sequence which encodes aZmMyb-IF35 sense RNA and a sequence which encodes a ZmMyb-IF35 antisenseRNA, and a linker which links the ZmMyb-IF35 sense RNA encoding sequenceto the ZmMyb-IF35 antisense RNA encoding sequence; and b) a promoter forregulating transcription of said DNA molecule, said promoter beingoperably linked to the ZmMyb-IF35 sense RNA encoding sequence and theZmMyb-IF35 antisense RNA encoding sequence.
 24. A method of preparing atransgenic plant containing altered levels of one or more phenoliccompounds, comprising a) transforming a plant cell with the expressionconstruct of claim 23; and b) regenerating a plant from said transformedplant cell.
 25. A transgenic plant prepared by the method of claim 24.